Serum Proteomic Profiling and Study of Haptoglobin Polymorphisms in Patients with Chronic GVHD after Allogeneic Hematopoietic Stem Cell Transplantation.

Abstract

We studied serum proteomic profiling in an attempt to discover novel biomarkers in the setting of clinically manifest graft versus host disease (GVHD) after allogeneic hematopoietic cell transplantation (Allo-HCT) by two-dimension gel electrophoresis (2-DE) and mass spectrometry analysis. More than 1000 protein spots per gel could be detected in individual samples and the distribution patterns of these spots differed significantly between the samples collected pre- and post- chronic GVHD (cGVHD) in the same patients. Mass spectrometric analysis in the paired 2DE gels from individual patients pre- and post-GVHD were performed. A group of proteins, Haptoglobin (Hp), Alpha-1-antitrypsin, Apolipoprotein A-IV, Serum paraoxonase and Zn-alpha-glycoprotein were demonstrated to be up-regulated and the proteins, Clusterin precursor, Alpha-2-macroglobulin, Serum amyloid protein precursor, Sex hormone-binding globulin, Serotransferrin and Complement C4 were found to be down-regulated in the patients with extensive cGVHD. Serum Hp was significantly increased in most patients following the development of cGVHD; Hp has been demonstrated to serve as an immuno-modulatory acute phase protein and may involve T- cell activation; further, Hp specific phenotypes have defined roles in influencing the pathophysiology of several immune system disorders. For these reasons, we used immunoblotting and PCR in combination with 2-DE gel image analysis to determine Hp polymorphisms in 24 allo-HCT patients (14 chronic GVHD, 4 acute GVHD and 6 without GVHD) and 10 normal donors. The results demonstrate that patients with cGVHD had a higher incidence of HP 2-2 phenotype (50.0%), in comparison to the patients without cGVHD (0%) and normal donors (10%) (Table 1), suggesting that the specific Hp polymorphism may play a role in the development of cGVHD after allo-HCT. A prospective analysis of Hp phenotype and the subsequent development of cGVHD is now being planned. This limited analysis suggests that proteomic profiling may provide significant contributions not only for identification of novel prognostic biomarkers, but also for novel insights into the pathophysiologic mechanisms of GVHD in allo-HCT.HP PhenotypePatients1-12-12-2Chronic GVHDn=142(14.3%)5(35.7%)7(50.0%)Acute GVHDn=4031no GVHDn=61(16.7%)5 (83.3%)0Normal Donorn=101(10.0%)8(80.0%)1(10.0%)